Non-fucosylated protein and methods thereof
Inventors
Chatterjee, Sohang • Rodrigues, Kavitha Iyer • Ghosh, Maloy • Maity, Sunit • Pendse, Rajeshwari • Unnikrishnan, Divya • M., Yogendra Manjunath B. • Arika, Jahnabi • M., Sathyabalan • M., Pavithra • Prasad, Bhargav • K., Veeresha • Pathak, Prabhat Kumar • Bhattacharjee, Sanghamitra • D., Pravin Kumar • Halan, Vivek • Srinivasan, Sankaranarayanan • Hora, Anuradha • N., Bairavabalakumar • Nair, Karthika • Thanigaivel, Aswini • Maliwalave, Amol • Shenoy, Bharath R. • Kurup, Anisha
Assignees
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Publication Number
US-10077434-B2
Publication Date
2018-09-18
Expiration Date
Abstract
The present disclosure relates to developing cell lines where specific biological pathways are modified, particularly by modifications in the enzymes of the cell. The present disclosure develops protein expression systems wherein specific modification of glycan chain of the protein is achieved, which produces non-fucosylated proteins, including non-fucosylated antibodies. The non-fucosylated proteins are used in developing therapeutic monoclonal antibodies and biomarkers, and in diagnosis and prognosis of various diseases. The present disclosure employs the Transcription Activator like Effector Nuclease (TALEN) technology for inactivating fucosylation in a cell.
Core Innovation
The disclosure describes TALEN-based inactivation of the FUT8 (α-1,6 fucosyltransferase) fucosylation pathway to generate fucose-knockout cell lines. The targeted outcome is production of afucosylated proteins, including afucosylated therapeutic monoclonal antibodies. The cell platform is implemented by introducing genome edits at a FUT8 target locus using TALEN DNA-binding domains and an endonuclease component.
DNA-binding domains are defined as TALE DNA-binding segments comprising one or more amino acid sequences selected from SEQ ID No. 6, SEQ ID No.8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 17, SEQ ID No.20, SEQ ID No. 23, SEQ ID No.26, SEQ ID No. 29, SEQ ID No.32, SEQ ID No.35, and SEQ ID No.38. The TALEN system binds to FUT8 gene exon-associated target sites, with an example targeting Exon 9. The nuclease component includes FokI, including Fok1.
Functional selection identifies FUT8 knockout clones using Lens Culinaris Agglutinin (LCA) and LCA-FITC flow cytometry to detect loss of fucosylated surface proteins. The disclosure further reports that genome edits yield INDELs/frameshifts/stop codons at the FUT8 target locus, supporting fucose knockout. Afucosylated antibodies produced on the edited cell platform are associated with enhanced ADCC.
Claims Coverage
The partial claim set provided includes one independent claim covering a TALE DNA-binding domain defined by a specified set of amino acid sequences. The inventive features are expressed as enumerated SEQ ID amino-acid sequences that form the DNA-binding domain of a TALE nuclease protein, with dependent claim coverage narrowing to binding specificity and particular nuclease and target locus constraints.
DNA-binding domain comprising selected TALE sequences
A DNA-binding domain of Transcription Activator like Effector Nuclease protein, wherein the DNA-binding domain comprises one or more amino acid sequence selected from the group consisting of SEQ ID No. 6, SEQ ID No.8, SEQ ID No. 11, SEQ ID No. 14, SEQ ID No. 17, SEQ ID No.20, SEQ ID No. 23, SEQ ID No.26, SEQ ID No. 29, SEQ ID No.32, SEQ ID No. 35, and SEQ ID No.38.
FUT8 gene sequence binding specificity using multiple selected SEQ IDs
A DNA-binding domain binds multiple Fut8 gene sequence regions, where each specified SEQ ID number binds a corresponding Fut8 gene sequence SEQ ID number.
Fok1 nuclease specification
The transcription activator-like effector nuclease protein wherein the nuclease is the Fok1 endonuclease.
Exon 9 targeting of Fut8 cleavage
Cleaving the Fut8 gene sequence that codes for the α-1,6 fucosyltransferase enzyme at Exon 9.
Across the provided independent and dependent claim excerpts, the coverage centers on enumerated TALE DNA-binding domain sequences that target FUT8 gene regions, including location-constrained cleavage at Exon 9. Coverage also includes specifying the nuclease as Fok1, and constraining binding relationships between selected TALE sequences and corresponding Fut8 gene sequence regions.
Stated Advantages
Enhanced ADCC for afucosylated therapeutic monoclonal antibodies.
Documented Applications
Producing afucosylated therapeutic monoclonal antibodies using a FUT8 fucose-knockout cell platform created with TALEN-based genome edits.
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